We have found that a transforming protein pp60src from chick embryo fibroblasts infected with a tumor avian sarcoma virus is located almost entirely in the cytoskeletal framework. Cytoskeletons were prepared by lipid extraction of transformed cells with a mild detergent and the transforming protein recognized by its protein kinase activity, by its precipitation with immune sera from tumor-bearing rabbits (TBR) and by the molecular weight (60,000) of the radioactive products on electropherograms. The pp60src of cytoskeletons also may be labeled in situ with (gamma 32P) ATP at a tyrosine site near the carboxyl terminus. We also are continuing to synthesize peptides from various regions of pp60src which may be functionally important for its kinase and transforming activities and are testing these peptides for their effects on these activities. These synthetic peptides are being used as immunogens for the generation of site-specific antibodies to identify src-related cellular proteins. To obtain a better understanding of the properties of pp60src which are important for transforming activity, we plan to construct site-directed deletion and point mutants of src and identify the activities of these altered src proteins in NIH 3T3 mouse cells. (R)